KINETIC-ANALYSIS OF THE ACTIVATION OF TRANSDUCIN BY PHOTOEXCITED RHODOPSIN - INFLUENCE OF THE LATERAL DIFFUSION OF TRANSDUCIN AND COMPETITION OF GUANOSINE DIPHOSPHATE AND GUANOSINE TRIPHOSPHATE FOR THE NUCLEOTIDE SITE

The activation of transducin (T) by photoexcited rhodopsin (R*) is kinetically dissected within the framework of Michaelis-Menten enzymology, taking transducin as substrate of the enzyme R*. The light scattering "release" signal (Vuong, T. M., M. Chabre, and L. Stryer. 1984. Nature (Lond.). 311:659-661.) was used to monitor the kinetics of transducin activation at 20-degrees-C. In addition, the influence of nonuniform distributions of R* on these activation kinetics is also explored. Sinusoidal patterns of R* were created with interference fringes from two crossed laser beams. Two characteristic times were extracted from the Michaelis-Menten analysis: t(form), the diffusion-related time needed to form the enzyme-substrate R*-transducin is 0.25 +/- 0.1 ms, and t(cat), the time taken by R* to perform the chemistry of catalysis on transducin is 1.2 +/- 0.2 ms, in the absence of added guanosine diphosphate (GDP) and at saturating levels of guanosine triphosphate (GTP). With t(form) being but 20% of the total activation time t(form) + t(cat), transducin activation by R* is not limited by lateral diffusion. This is further borne out by the observation that uniform and sinusoidal patterns of R* elicited release signals of indistinguishable kinetics. When [GDP] = [GTP] = 500-mu-M, t(cat) is lengthened twofold. As the in vivo GDP and GTP levels are comparable, the exchange of nucleotides may well be the rate-limiting process.